Endothelial Barrier Antigenimmunoreactivity is Conversely Associated with Blood-Brain Barrier Dysfunction after Embolic Stroke in Rats

While the concept of the Neurovascular Unit (NVU) is increasingly considered for exploring mechanisms of tissue damage in ischemic stroke, immunohistochemical analyses are of interest to specifically visualize constituents like the endothelium. Changes in immunoreactivity have also been discussed to reflect functional aspects, e.g., the integrity of the blood-brain barrier (BBB). This study aimed to characterize the endothelial barrier antigen (EBA) as addressed by the antibody SMI-71 in a rat model of embolic stroke, considering FITC-albumin as BBB leakage marker and serum levels of BBB-associated matrix metalloproteinases (MMPs) to explore its functional significance. Five and 25 h after ischemia onset, regions with decreased BBB integrity exhibited a reduction in number and area of EBA-immunopositive vessels, while the stained area per vessel was not affected. Surprisingly, EBA content of remaining vessels tended to be increased in areas of BBB dysfunction. Analyses addressing this interrelation resulted in a significant and inverse correlation between the vessels’ EBA content and degree of BBB permeability. In conclusion, these data provide evidence for a functional relationship between EBA-immunoreactivity and BBB dysfunction in experimental ischemic stroke. Further studies are required to explore the underlying mechanisms of altered EBA-immunoreactivity, which might help to identify novel neuroprotective strategies.Key words: ischemic stroke, embolic model, EBA, blood-brain barrier, matrix metalloproteinase     

Perineuronal Nets Characterized by Vital Labelling, Confocal and Electron Microscopy in Organotypic Slice Cultures of Rat Parietal Cortex and Hippocampus

Perineuronal nets (PNs) of the extracellular matrix have been shown to develop in organotypic slice cultures largely corresponding with regional patterns known from in vivo experiments. In the present study, we use vital labelling to investigate aspects of the cell type-dependent development of PNs associated with nonpyramidal neurons and pyramidal cells in the parietal cortex and hippocampus. Frontal sections were cut from brains of 3-5-day-old rats and were cultured for 3-5 weeks. PNs were sequentially labelled using biotinylated Wisteria floribunda agglutinin and chromogen-tagged streptavidin either in living slice cultures, examined by confocal microscopy in vitro, or in cultures examined by confocal and electron microscopy after fixation. Nonpyramidal and pyramidal cells were characterized by immunoreaction for parvalbumin and the ionotropic glutamate receptor subunits 2/3. Vital labelling and examination of fixed slices correspondingly revealed that large numbers of PNs developed around cortical and hippocampal interneurons under depolarizing conditions induced by elevated external potassium concentration. After culture in standard medium, PNs were mainly found in association with subpopulations of pyramidal cells in the parietal cortex. PNs showed ultrastructural characteristics resembling those known from perfusion-fixed brain. A zone of labelled extracellular matrix aggregates was found in close proximity to the neuronal cell surface, surrounding presynaptic boutons and preterminal axons. The results show that characteristic features of PNs are retained after vital labelling in slice cultures. Moreover, our findings suggest that the cell type-specific development of PNs is regulated by patterns of intrinsic activity mediated by intra-cortical and -hippocampal synaptic contacts on potentially net-associated neurons.     

Changes of the organotypic retinal organization in Borna virus-infected Lewis rats

Retinae of Borna disease virus (BDV)-infected Lewis rats were investigated with emphasis on long-term changes in organotypic tissue organization and glia-neuron relationship. Virus inoculation was attained via intracerebral BDV injection. Following survival times ranging between two and eight months, the retinal thickness was reduced up to one third of that of controls. Photoreceptor segments were completely extinguished and the number of neurons was dramatically reduced. The typical laminar organization of the retina was largely dissolved. Electron microscopy revealed severe spongy degeneration. Large numbers of activated microglia and macrophages were found, both cell types performing very active phagocytosis. The microglial cells expressed an extraordinary phenotype as characterized by large numbers of processes, with some of them penetrating the endfeet of Müller cells and others establishing highly complex interdigitations with vacuolized swellings and endings of neuronal processes. Müller cells were not reduced in number but displayed clear indications of gliosis such as alterations in the immunoreactivity for filament proteins and glutamine synthetase, significantly thickened stem processes, and an altered pattern of K⁺ currents in patch-clamp recordings. These findings demonstrate for the first time long-term neuron-glia interactions in the retina of BDV-infected rats. Moreover, the data contribute to our knowledge on structural and functional alterations accompanying persisting virus infection in the central nervous system.     

Spatio-temporal course of macrophage-like cell accumulation after experimental embolic stroke depending on treatment with tissue plasminogen activator and its combination with hyperbaric oxygenation

Inflammation following ischaemic stroke attracts high priority in current research, particularly using human-like models and long-term observation periods considering translational aspects. The present study aimed on the spatio-temporal course of macrophage-like cell accumulation after experimental thromboembolic stroke and addressed microglial and astroglial reactions in the ischaemic border zone. Further, effects of tissue plasminogen activator (tPA) as currently best treatment for stroke and the potentially neuroprotective co-administration of hyperbaric oxygen (HBO) were investigated. Rats underwent middle cerebral artery occlusion and were assigned to control, tPA or tPA+HBO. Twenty-four hours, 7, 14 and 28 days were determined as observation time points. The accumulation of macrophage-like cells was semiquantitatively assessed by CD68 staining in the ischaemic area and ischaemic border zone, and linked to the clinical course. CD11b, ionized calcium binding adaptor molecule 1 (Iba), glial fibrillary acidic protein (GFAP) and Neuronal Nuclei (NeuN) were applied to reveal delayed glial and neuronal alterations. In all groups, the accumulation of macrophage-like cells increased distinctly from 24 hours to 7 days post ischaemia. tPA+HBO tended to decrease macrophage-like cell accumulation at day 14 and 28. Overall, a trend towards an association of increased accumulation and pronounced reduction of the neurological deficit was found. Concerning delayed inflammatory reactions, an activation of microglia and astrocytes with co-occurring neuronal loss was observed on day 28. Thereby, astrogliosis was found circularly in contrast to microglial activation directly in the ischaemic area. This study supports previous data on long-lasting inflammatory processes following experimental stroke, and additionally provides region-specific details on glial reactions. The tendency towards a decreasing macrophage-like cell accumulation after tPA+HBO needs to be discussed critically since neuroprotective properties were recently ascribed to long-term inflammatory processes.     

Basic structural and functional characteristics of the epidermal barrier in wild mammals living in different habitats and climates

Based on the combination of standard light and transmission electron microscopy, cryo-SEM, immunohistochemistry and a new sensitive glycolipid histochemical technique (5-hexadecanoylaminofluorescein staining, laser scanning microscopy), including densitometrical evaluation, our approach gives for the first time an overview of the specific biology of the epidermal permeability barrier in wild mammals (20 species from five orders), living under varying (aquatic or moist to dry) habitat conditions. The results obtained emphasised that the barrier region in most of the species studied is a continuous zone (thickness, 0.1 and 3 μm) between the upper cells of the stratum granulosum and the inner cells of the stratum corneum conjunctum, normally present as a homogeneous glycolipid layer originating from fusion of lamellar body contents after exocytotic activities of the granular cells. However, this finding did not apply to all of the species studied, i.e., the Wild boar, the Common seal and the three large species with a very thick vital epidermis, the African elephant, the hippopotamus and the common dolphin, exhibited variations from the basic scheme. Densitometric evaluation of the 5-hexadecanoylaminofluorescein staining revealed that reaction intensity was not only generally related to the habitat conditions but also to vital epidermis thickness and hair density. The immunohistochemical demonstration of Na⁺/H⁺ exchanger 1 corroborated for all wild mammals studied that this important regulator of pH conditions during barrier formation is continuously produced in the epidermis. The variations in barrier biology observed for some species obviously had to be developed in relation to animal size (or body size area) and hair coat density, but, particularly, by the specific adaptation of certain mammalian groups to the aquatic environment. In the latter case, the typical barrier zone system was lost, as in the hippopotamus or the cetaceans.     

Infrageneric variability of pollen morphology in Palaua (Malveae,Malvaceae) and the taxonomic utility of quantitative pollen characters

Palaua (Malvaceae) comprises 15 species endemic to the coastal deserts of Chile and Peru. Previous molecular phylogenetic analyses have shown that this genus is monophyletic and can be subdivided into three clades. In the present study, pollen morphology of all species of Palaua was examined using light and scanning electron microscopy to determine whether it provides additional data in support of the proposed infrageneric groups. The pollen grains are spheroidal, medium to large in diameter, spinose and pantocolporate – the ectocolpi are very short. The tectum is perforate and characterised by granula. In all species the nexine is of a similar thickness in all areas of the pollen grain, while the sexine thickness varies, being notably thicker in the areas where the broad spine bases are sited (‘spine cushions’). Many of the quantitative characters have conspicuous variability, but the variability shows considerable overlap between species. Nonetheless, P. guentheri, P. inconspicua, P. malvifolia and P. modesta are united as a group by having the smallest pollen grains with the smallest apertures, the shortest spines and the shortest interspinal distance. This grouping reflects only partially the suggested infrageneric clades, although it does tend to unite the species with the smallest flowers. A possible link between reproductive resources and pollen size is also considered, as well as the influence of polyploidy. However, the taxonomic utility of these quantitative characters is weakened by species with more than one cytotype.     

Retinal pigment epithelium melanin granules are phagocytozed by Müller glial cells in experimental retinal detachment

The ability of retinal Müller glial cells to perform phagocytosis in vivo is studied in a rabbit model of experimental retinal detachment where pigment epithelial cells are occasionally detached together with the neural retina. While macrophages and/or microglial cells phagocytoze most of the cellular debris at the sclerad surface of the detached retinae, some Müller cells accumulate melanin granules. The granules are virtually intact at the ultrastructural level, and are surrounded by a membrane. They are often located close to the sclerad end of the cells, but some are distributed throughout the outer stem process up to the soma. It is concluded that rabbit Müller cells in vivo are capable of phagocytosis and of transporting the phagocytozed material within their cytoplasm.